1. Field of the Invention
This invention relates to methods and apparatus for separating blood and to processes for producing contact-activating agents adapted to activate clotting factors in blood. More specifically this invention relates to methods and apparatus for separating blood which utilize powdered contact-activating agents to activate blood clotting and to processes for making powdered contact-activating agents.
2. Prior Art
It is well known that the in vitro coagulation of human blood involves a series of enzymatic activation processes and that these processes are enhanced by contacting blood against certain foreign surfaces, especially silicaeous substances, such as glass, kaolin, bentonite, hydrated aluminum silicate and diatomaceous silica or Kieselguhr. A study of the role of surface contact with these materials was published in 1960 the British Journal of Haematology, volume 6, pages 88 through 101, in an article by J. P. Soulier and O. Prou-Wartelle entitled, "New Data on Hageman Factor and Plasma Thromboplastin: The Role of `Contact` in the Initial Phase of Blood Coagulation".
Blood separation practices have long utilized the clot activation properties of glass insofar as glass tubes have been used to collect and separate blood. The standard method for separating blood has been, first, to deposit a predetermined quantity of blood into a glass tube; second, activating the clotting factors within the blood at the inner surface of the tube; third, without agitation, permitting the activated clotting factors to diffuse or migrate toward the center of the tube; fourth, maintaining the clotting process to obtain substantially complete coagulation; and fifth, centrifugally separating the coagulated blood into a lighter phase consisting essentially of serum and a heavier phase consisting essentially of cellular and fibrillar matter.
In order to assure substantially complete coagulation, this standard method normally involves a lengthy waiting period between the collection or deposition of the blood into the tube and the centrifugal separation of the blood. In fact this waiting period is no less than 30 minutes and may last as long as 100 minutes, the length of time being dependent upon the clotting characteristics of the particular blood sample. It has been theorized that the clotting time is dependent on the total surface area of the glass and the duration of time that the blood is in contact with the surface area. I have found that the clotting time is also dependent upon the rate of diffusion or migration of the activated clotting factor from the activation sites, that is, at the inner surface of the conventional blood separation vessel to the center of the vessel.
Thus a major problem associated with the standard blood separation methods and practices is the lengthy waiting period needed to assure substantially complete coagulation before separation. It is an object of this invention to provide a method and apparatus for separating blood which substantially reduces this waiting period.
Another problem associated with the standard blood separation methods and practices relates to the latent formation of fibrin in the lighter or serum portion of the separated blood. The blood coagulation process includes the conversion of fibrinogen to fibrin, fibrin being the fibrillar structure within which cellular material (red cells, white cells, platelets, debris, etc.) are entrapped to form a clot. In the event that a blood sample is centrifugally separated before coagulation is substantially complete, the lighter or serum portion will contain fibrinogen and activation agents which will react in the separated lighter portion to form fibrin strands. Inasmuch as the testing devices used to analyze the serum portion include intricate plumbing systems, it is essential that the serum portion be free from fibrin contamination. Also, it should be noted that even minute amounts of fibrin contamination of the serum might affect the apparent chemistries of the serum measured by the testing devices, should the amount of fibrin be so minute as to pass through the testing device without obstructing the passageways therein.
Accordingly, another object of this invention is to provide methods and apparatus which will eliminate the problem of latent fibrin formation within the lighter or serum portion of a separated blood sample.
Another problem associated with standard blood separation devices and methods pertains to the relative cleanliness of the lower portion of the serum or lighter portion of separated blood. After complete coagulation, the clot of cellular and fibrillar matter extends throughout the volume of the coagulated blood. Upon centrifugation, due to the higher specific gravity of the cellular portion of the blood, the cellular matter is sedimented under the influence of the applied centrifugal force, and the fibrillar matter is drawn with the cellular matter out of the lighter portion. Fibrillar strands in the upper portion of the pre-centrifuged blood may be unattached to cellular portions; in this event, these strands may remain suspended in the serum portion after centrifugation. Also, such strands may be free from red cells but attached to the fibrin network of the precentrifuged clot, in which event such strands may, after centrifugation, remain attached to the clotted material but remain suspended above the bulk of the heavier portion of the separated sample. Thus, a fibrin or "white layer" may appear at the interface between separated portions of the blood. In order to avoid contamination of the serum with this matter, less serum may be aspirated from the separated blood than actually is available in the lighter portion of the blood.
Accordingly it is another object of this invention to provide an apparatus and method for separating blood which eliminates this problem of fibrin contamination. More specifically, it is an object of this invention to eliminate fibrin contamination caused by either latent conversion of fibrinogen into fibrin after centrifugation or by the suspension within the serum portion of fibrin formed before centrifugation.
It is another important object of this invention to provide methods and apparatus for separating blood which permit the utilization of separation vessels made from materials which are durable, safe, convenient to use, inexpensive and easy to manufacture in any form, and yet which would not require skills or manipulations in excess of those required for standard blood separation procedures. The standard vessel used in separating blood is made from glass; and coagulation is accomplished without agitation, with the inner surface of the glass being the only surface used to activate the clotting factors. The present invention provides for a method of activating the clotting factors at surfaces other than those of the vessel and thus permits the utilization of vessels made from plastic, metal and other materials, without additional manipulative steps beyond those required under conventional procedures for blood separation.
A more recently developed method for separating blood utilizes glass or kaolin granules within a plastic tube. In this method, the granules are deposited in the tube, blood is supplied to the tube, and the tube is agitated by a mixing device. In addition to the activation of clotting factors at the inner surface of the tube, activation also takes place at the surfaces of the granules.
However, this method requires mechanical agitation immediately after the collection of the blood and throughout at least the initial stages of coagulation of the blood. Without such mechanical stirring, the granules would remain in their initial positions within the filled tube, generally adjacent the tube wall and thus would activate only the peripheral bottom portions of the blood within the tube.
Accordingly, it is another important object of this invention to provide apparatus and methods for separating blood which do not require agitation of the blood to be separated.